Genetic characterization and molecular survey of Babesia sp. Xinjiang infection in small ruminants and ixodid ticks in China

Highlights

• The Babesia sp. Xinjiang in blood and tick samples were first studied from a broad geographic range in China using PCR.

• The Babesia sp. Xinjiang infection in H. longicornis was first reported in the present study.

• The sequences of rap-1a gene of Babesia sp. Xinjiang indicated 100% identity between sheep/goat and ticks.

Abstract

Babesia sp. Xinjiang is a large ovine Babesia species that was recently isolated in China. Compared with other ovine Babesia species, it has different morphological features, pathogenicity and vector tick species. The known transmitting vector is Hyalomma anatolicum. In this study, the distribution and the presence of Babesia sp. Xinjiang in small ruminants and ixodid ticks in China were assessed by specific nested-PCR assay based on the rap-1a gene. A total of 978 blood samples from sheep or goats from 15 provinces and 797 tick specimens from vegetation from 10 provinces were collected and analysed for the presence of the Babesia sp. Xinjiang. Full-length and partial rap-1a of Babesia sp. Xinjiang were amplified from field samples. The PCR results were further confirmed by DNA sequencing. Overall, 38 (3.89%) blood samples and 51 (6.4%) tick samples were positive for Babesia sp. Xinjiang infection. The highest presence (26.92%) was found in blood samples from Yunnan province, while H. qinghaiensis ticks with the highest presence of infection (21.3%) were from Gansu province. This study identified for the first time Babesia sp. Xinjiang infection in H. longicornis tick species. The rap-1a sequences of Babesia sp. Xinjiang from field blood and tick samples indicated 100% identity. The presence of Babesia sp. Xinjiang infection may increase in China. Novel potential transmitting vectors might be more extensive than previously thought.

Introduction

Babesia sp. Xinjiang is a sheep Babesia species, only distributed in China. This parasite is isolated from sheep experimentally infected with Rhipicephalus sanguineus and Hyalomma anatolicum, which are collected from farmed sheep and goats in Kashi region, Xinjiang Uygur Autonomous Region in northwestern China (Guan et al., 2001). It is a large parasite with various morphological forms in infected erythrocytes: single and paired piriform are the most common shapes. The known vector is H. anatolicum (Guan et al., 2009).

The pathogenic and morphological characteristics of Babesia sp. Xinjiang are different from those of B. motasi (European isolates), B. ovis and B. crassa, which have been found in small ruminants worldwide, and Babesia cf. motasi in China (Guan et al., 2009, Liu et al., 2007). Molecular taxonomy and phylogenetic reconstruction based on 18S rDNA gene and ITS rDNA confirmed these biological differences, placing Babesia sp. Xinjiang in a clade clearly separated from all other small ruminant Babesia species described thus far (Liu et al., 2007, Niu et al., 2009). Babesia sp. Xinjiang is phylogenetically closely related to other Babesia species recently described from wild ruminants in South Africa and to B. pecorum isolated from red deer in Spain (Oosthuizen et al., 2009, Jouglin et al., 2014).

Currently, the rap-1 gene is being used as a marker for the molecular diagnosis of B. bovis (Silva et al., 2009, Molad et al., 2015) and B. bigemina in bovine babesiosis (Hilpertshauser et al., 2007, Niu et al., 2015a) and Babesia cf. motasi isolates from ovine babesiosis in China (Niu et al., 2016a). In Babesia sp. Xinjiang, 7 different rap-1a gene copies were previously described, and the 5′ regions exhibited identical sequences of 936 nt in all 7 rap-1a gene copies, the 3′ regions differed at 28 positions in 147 nt (Niu et al., 2015b). The conserved 5′ region of the multiple rap-1a gene copies in Babesia sp. Xinjiang may ensure that all rap-1a copies are amplified and thus improving parasite detection. Specific PCR assays targeting the multiple copies of rap-1a could be developed to detect rap-1a genes, even when Babesia sp. Xinjiang DNA concentration is low in field samples. It could therefore be used for epidemiological investigation in the field.

The molecular detection of ovine babesiosis caused by Babesia sp. Xinjiang in vertebrate hosts and in ixodid ticks, and the genetic diversity of Babesia sp. Xinjiang between the ticks and vertebrate host in different regions have been poorly studied in China (Guan et al., 2008, Omar Abdallah et al., 2016). In this report, genomic DNA samples extracted from field blood samples and questing ticks from different regions were amplified based on the full-length (outer) and 5′ region (inter) of rap-1a using specific nested-PCR assays to test for the presence of Babesia sp. Xinjiang. The presence of Babesia sp. Xinjiang in different regions of China was investigated. The sequences of the 5′ region of rap-1a obtained from positive Babesia sp. Xinjiang DNA samples were analysed, and the genetic diversity of rap-1a among different isolates from different regions was characterized and discussed.